Recombinant influenza a viruses

ABSTRACT

The invention relates to a recombinant NS gene of an influenza A virus comprising a functional RNA binding domain and a gene sequence modification after nucleotide position 400 of the NS1 gene segment, counted on the basis of influenza A/PR/8/34 Virus, wherein the modification bars transcription of the remaining portion of the NS1 gene segment. It further relates to embodiments, wherein the modification comprises deletions, insertions, or a shift of the open reading frame, and particularly to constructs comprising an insertion of an autocleavage site 2A, the nef gene from HIV-1 or the sequence encoding the ELDKWA-epitope of gp4l of HIV-1. The invention also relates to influenza virus transfectants that contain the modified NS gene and have an IFN inducing phenotype but which may or may not be sensitive towards IFN. The invention also relates to vaccines comprising such a chimeric virus.

TECHNICAL FIELD

[0001] The invention is in the fields of vaccine development andapplication and relates to attenuated live vaccine vectors, morespecifically to such vectors based on or derived from geneticallymodified influenza A virus strains, and to the manufacture of recominantinfluenza viruses and vaccines.

BACKGROUND OF THE INVENTION

[0002] Influenza viruses are segmented negative-strand RNA viruses andbelong to the Orthomyxoviridae family. Influenza A virus consists of 9structural proteins and codes additionally for one nonstructural NS1protein with regulatory functions. The non-structural NS1 protein issynthesized in large quantities during the reproduction cycle and islocalized in the cytosol and nucleus of the infected cells. Thesegmented nature of the viral genome allows the mechanism of geneticreassortment (exchange of genome segments) to take place during mixedinfection of a cell with different viral strains. Several features makeinfluenza viruses attractive candidates for the development of effectivelive vaccine vectors against different diseases:

[0003] (i) influenza viruses induce strong cellular and humoral immuneresponses, at the systemic and the mucosal level against viral proteinsfollowing infection;

[0004] (ii) influenza virus as an RNA virus does not contain a DNA phasein its replication cycle. Therefore chromosomal integration of viralgenes into the host can be excluded;

[0005] (iii) many different influenza virus subtypes are available.Since antibodies against these different subtypes show no or littlecrossreactivity, pre-existing immunity to the viral vector in the host,which is frequently a problem for other live vectors, can becircumvented. Also, effective booster immunizations with differentsubtype influenza viruses expressing the same antigens might bepossible; and

[0006] (iv) attenuated influenza viruses as live influenza vaccines,which were shown to be safe and immunogenic in humans, are available.

[0007] Until now the main problem of utilizing influenza virus as avector concerns the size of the virus genome and its limited capacity totolerate foreign sequences. Among ten influenza viral proteins, only thesurface glycoproteins haemagglutinin (HA) and neuraminidase (NA) havebeen successfully engineered for stable expression of foreign epitopes.Since influenza virus tolerates an insertion of only approximately 10amino acids into its HA molecule, there is only a limited possibility toinfluence the conformation properties of inserted epitopes which wouldprobably be better presented if longer sequences would be introduced.Besides, surface influenza glycoproteins such as HA or NA cannot beconsidered as optimal targets for the presentation of foreign sequencesbecause of their association with antigenic properties of the viruses.An HA live virus construct containing a desired foreign antigen is notapplicable for boosting immunizations (e.g., by second and furtheradministrations) because of the pre-existing immunity against the HAcaused by the first immunization or by a natural virus infection. Abooster immunization would be possible only upon introduction of thedesired antigenic structure into another HA molecule belonging to adifferent influenza virus subtype. It is evident that such a process isdifficult, laborious and extremely time consuming and therefore unlikelyto be suitable for routine vaccine preparation

[0008] Preceding investigations in connection with the present inventionhave indicated that the NS gene of influenza A virus may be a promisingalternative to HA as a viral carrier for presenting a desired foreignantigen to the animal or human immune system. The recently establishedmethod of reverse genetics (Egorov et al., 1998, J Virol 72(8), 6437-41)allows to rescue influenza viruses containing long deletions orinsertions of foreign sequences at the carboxyl side of thenon-structural Protein 1 (NS1 protein). NS1 protein is abundant ininfluenza virus-infected cells and stimulates cytotoxic T-lymphocyte(CTL) responses as well as antibody responses during the natural courseof influenza virus infection.

[0009] Further details about the influenza virus NS gene can be found inWO 99/64571. Additionally, WO 99/64571 discloses that attenuatedinfluenza A virus transfectants containing knockout deletions of theentire NS1 gene were found to have a strong interferon (IFN) inducingphentopye. This was concluded from the finding that such transfectantswere able to grow on IFN deficient Vero cells but were unable to grow onhen eggs or Madin-Darby Canine Kidney (MDCK) cells.

[0010] The influenza NS1 protein is an RNA-binding protein which hasbeen implicated in a number of regulatory functions during influenzavirus infection.

[0011] It is synthesized in large amounts and found mainly in thenucleus early during infection and later in the viral cycle in thecytoplasm of the infected cells. Other than the influenza NS-1 protein,another regulatory viral protein, namely the Nef protein of HIV-1 whichis a myristylated protein, is localized in the cytosol in associationwith the cell membrane.

[0012] An immune response directed against early expressed regulatoryHIV-1 proteins could possibly allow the elimination of virus-infectedhost cells in the replication cycle before release of new infectiousviral particles would even occur. As the Nef protein is among the firstones to be released and further is one of the major HIV proteinsproduced following infection, it could play a crucial role in developingan efficacious anti-AIDS vaccine.

[0013] The HIV-1 “negative factor” (Nef) is encoded by an open readingframe which is located at the 3′ end of the virus, partially overlappingthe U3 region of the 3′ long terminal repeat. Up to 80% of the early,multiply spliced class of viral transcripts encode Nef. The Nef geneproduct is an NH₂-terminally myristylated protein of 27 to 30 kDa, whichis predominantly localized in the cytoplasm and associated with themembrane and the cytoskeletal matrix. It is well conserved among thedifferent human (HIV-1 and HIV-2) and Simian immunodeficiency viruses(SIV).

[0014] The close evolutionary relationship between these primatelentiviruses suggests that the Nef protein plays an important role inviral infection and pathogenesis, although the exact role in the viruslife cycle and its functions at the cellular level are still the subjectof current research.

[0015] Various details about the Nef protein and its effects are alreadyknown, however. For instance, it is reported that some humans infectedwith Nef-deleted, HIV remained disease-free, with normal CD4 counts 10to 14 years after infection, although deletion of Nef is not a universalfinding in long-term nonprogressors. In addition, Nef-deficient SIVfails to produce AIDS in infected adult macaques. SIV mutants deletedfor the Nef gene even induce protection against a virulent challenge.Nef was shown to stimulate HIV-1 proviral DNA synthesis and itsexpression has also been found to induce the efficient internalizationand degradation of the cell surface CD4 receptor for HIV-1. ThisNef-induced CD4 down-regulation, which renders cells resistant to viralsuperinfection, has the potential to increase virus replication byfacilitating release of progeny virions. It was further demonstratedthat extracellular Nef protein could activate HIV-1 from latent toproductive infection both in infected T-cell lines and in PBMC fromasymptomatic carriers. Further, it was shown that CTLs inefficientlylysed primary cells infected with HIV-1, if the viral Nef gene productwas expressed.

[0016] Protection of HIV-infected cells from efficient recognition andkilling by CTLs correlates with the Nef-mediated down-regulation of MHCclass I molecules. Nef also interferes with the induction of IL-2 mRNAin T-cell lines. Furthermore, there are a large number of cellularpartners that have been found to be associated with Nef expressionincluding Src family kinases, β-COP, a serine-threonine kinase,thioesterase and p53.

[0017] It is also reported that the majority (about ⅔) of HIV-1seropositive patients generated Nef-specific CTLs.

[0018] Two central multirestricted immunodominant regions (amino acids66 to 100 and 115 to 146) and a carboxyl-terminal region (amino acids182 to 206) were identified within the Nef protein. These threemultirestricted immuno-dominant regions (amino acid sequences containingmore than one T-cell epitope) are being recognized by human CD8+CTLs inassociation with at least 14 different MHC class I molecules includingthe important MHC haplotypes HLA-A1, -A3, -A11, -B8, -B17, -B18 and-B37.

[0019] The two central multirestricted domains of the Nef protein arethe most highly conserved regions among different HIV-1 isolates andwere imrnunodominant for most of the asymptomatic HIV-1 seropositivedonors tested. In addition to the high immunogenicity of the HIV-1 Nefprotein, which has been demonstrated by its capacity to induce strongT-cell immune responses, also Nef-specific B-cell immune responses arereported in the literature.

SUMMARY OF THE INVENTION

[0020] The present invention relates to the development of attenuatedlive vaccine vectors, more specifically to such vectors based on orderived from genetically modified influenza A virus strains. It furtherrelates to the construction and modification of genetically engineerednon-structural genes of influenza A viruses, particularly of the NS1gene segment, wherein the modifications include deletions of selectedparts of the NS1 gene segment and/or insertions of heterologous,preferably antigenic, sequences into selected sites of the NS1 gene. Itis another objective of the invention to provide chimeric influenzaviruses containing such modified NS1 gene segments but which do notsuffer from the drawback of being IFN sensitive, in contrast to thetransfectants disclosed in WO 99/64571. The invention further relates torecombinant proteins obtained from the NS1-modified viruses byexpression in a suitable host system and further to a vaccine comprisingthe NS1-modifed viruses of the present invention.

[0021] It is yet another objective of the invention to provide a methodfor obtaining recombinant influenza viruses as well as attenuatedinfluenza vaccines, based on the generation of continues cell lines(e.g. Vero, MDCK etc.) expressing synthetic influenza genes (minus senseRNA) comprising natural or engineered influenza sequences (deletions orinsertions). These cell lines producing high quantities of such genescan be used for infection with influenza virus followed by selectionprocedures in order to get a gene of interest incorporated into theviral progeny.

[0022] The present inventors have established a reverse genetics systemon Vero cells allowing them to manipulate the virulence of the PR8influenza A virus strain by changing the length of the translated NS1protein. In the course of the research leading to the present inventionthe capacity of influenza A virus to tolerate and present longinsertions in the NS gene have been investigated. A collection ofseveral chimeric NS1 gene constructs using heterologous sequencesincluding the HIV-1 derived sequences encoding ELDKWA of gp41 or Nef,has been established by insertion of one or more of the heterologoussequences or, optionally, several repeats of any such sequence, in frameinto the NS1 protein.

[0023] The aforementioned heterologous sequences were inserteddownstream nt position 400 (corresponding to aa position 124) andoptionally preceded by the 2A autocleavage site sequence and/or by aleader sequence derived from the influenza HA molecule. Other constructsadditionally comprised an anchor sequence derived from the influenza HAmolecule as an insertion right after the desired antigenic sequence(s),thus forming the end of the entire heterologous insertion. In each casethe insertions were followed by a stop codon to prevent transcriptionand translation of the remaining portion of the NS1 gene segment(including the effector domain), while maintaining the cleavage site forthe NS splicing (necessary for transcription and translation of theNS2=NEP gene segment) fully functional.

[0024] Rescued viruses caused expression and accumulation of the foreignantigens in the cytosol and/or on the surface of the infected cells. Theinventors also successfully rescued transfectant viruses harbouring amultirestricted immunodominant region rich in T-cell epitopes of HIV-1Nef protein (136 amino acids).

[0025] All transfectants displayed normal growth characteristics in Verocells, embryonated chicken eggs and MDCK cells, but were attenuated inmice. Chimeric influenza NS1-Nef viruses did not replicate inrespiratory tracts of infected mice, but were able to induce a strongNef-specific CTL response following a single intranasal immunization. Inaddition, a Nef-specific antibody response was detected following threeimmunizations. Transfer of the recombinant NS-nef gene by geneticreassortment from the viral PR8 (influenza A/PR/8/34; Egorov et al.,1994, Vopr. Virusol. 39:201-205) vector to other influenza strainsresulted in the same level of attenuation and immunogenicity. Thisfinding permitted the present inventors to perform effective boostingimmunizations using several attenuated vectors of different antigenicsubtypes.

[0026] Thus, the inventors were able to demonstrate that the approach tocreate a set of influenza chimeric strains belonging to differentinfluenza subtypes while bearing the identical recombinant chimeric NS1gene, gives the opportunity to create several strains for boostingimmunizations. They have further proven that once a new chimeric NS1gene construct is rescued it can be routinely transferred to anotherinfluenza strain by genetic reassortment.

DESCRIPTION OF THE FIGURES

[0027]FIG. 1 shows a functional map of the engineered NS1 protein ofPR8;

[0028]FIG. 2 shows the structure of recombinant NS1 proteins of therescued influenza transfectant viruses expressing gp-41 and IL-1peptides; FIG. 3 shows the structure of recombinant NS1 protein of therescued influenza transfectant PR82Anef (PR8/Nef) expressing aa7O-206 ofthe Nef protein of HIV-1 NL4-3;

[0029]FIG. 4 shows the replication of chimeric influenza/Nef viruses inmouse lower respiratory tracts;

[0030]FIG. 5 shows the number of IFN-gamma secreting cells fromimmunized mice per 10⁶ spleen cells detected after immune spleen cellswere incubated in the presence of Nef peptide, NP peptide or withoutpeptide as an indicator for T-cell responses;

[0031]FIG. 6 shows the number of IFN-gamma secreting cells from thelymph nodes draining the respiratory tracts from immunized mice per 10⁶spleen cells detected after immune spleen cells were incubated in thepresence of Nef peptide, NP peptide or without peptide as an indicatorfor T-cell responses;

[0032]FIG. 7 shows Nef-specific serum IgG immune responses following2^(nd) and 3^(rd) Immunizations of mice immunized with recombinantinfluenza/Nef viruses;

[0033]FIG. 8 exhibits results of a plaque reduction assay;

[0034]FIG. 9 exhibits results of an interferon induction assay;

[0035]FIG. 10a-10 c show immunofluorescence of Vero cells infectedpreviously with recombinant PR8/Nef virus (10 a, 10 b) and the wild typeinfluenza PR8 virus (10c).

[0036]FIG. 11 shows Nef peptide-specific and NP peptide-specific immuneresponses as a quantification of IFN-γ secreting cells in murine spleencells.

[0037]FIG. 12 shows Nef peptide-specific and NP peptide-specific immuneresponses as a quantification of IFN-γ secreting cells in lymph nodesdraining the respiratory tracts of immunized mice.

[0038]FIG. 13 shows Nef peptide-specific and NP peptide-specific immuneresponses as a quantification of IFN-γ secreting cells in urogenitalsingle cell populations of immunized mice.

[0039]FIG. 14 exhibits results of an ELISA assay determiningNef-specific IgG in sera of mice two weeks after the third immunizationwith the PR8/NS-Nef or the Aichi/NS-Nef vector.

[0040]FIG. 15 is a schematic representation of an Influenza NStranscription system for expression of minus sense RNA for use ingenerating recombinant influenza viruses.

DETAILED DESCRIPTION OF THE INVENTION

[0041] In one embodiment, the invention relates to geneticallyengineered NS gene constructs of an influenza A virus comprisingsequence modifications, i.e. deletions or insertions, between nucleotide(nt) positions 400 and 525 of the NS1 gene segment (numbering is basedon the NS gene of influenza A/PR/8/34 virus). Unexpectedly, it turnedout that maintaining functionality of the NS1 gene segment up to ntposition 400 (corresponding to aa position 124 of the NS1 protein) whileconcomitantly deleting the remaining portion or at least a major partthereof or inserting a foreign nt sequence into the region after ntposition 400 or shifting the reading frame to cause wrong transscriptionand translation of the remaining NS1 portion resulted in the rescue ofNS gene constructs that rendered their viral vectors IFN inducing butnot IFN sensitive.

[0042] This surprising finding was confirmed by experiments wherein thechimeric influenza viruses engineered according to the present inventionnot only induced a strong IFN response in MDCK cells and hen eggs butwere also able to grow on these host substrates at an efficiencycomparable to the wildtype PR8 virus. In contrast, chimeric influenzaviruses containing deletions of the first third of the NS1 gene ordeletions of the entire NS1 gene displayed an IFN inducing as well as anIFN sensitive phenotype. They were unable to grow on hen eggs or MDCKcells and therefore could only be cultivated on IFN deficient cell linessuch as Vero cells. Chimeric influenza viruses of the latter type havebeen disclosed in WO 99/64571. It is assumed that the viruses of thepresent invention, which are not as strongly attenuated as the virusesof WO 99/64571, are more immunogenic and therefore better suitable forthe manufacture of highly effective live vaccines against various kindsof viral infections.

[0043] In another embodiment of the invention the genetically engineeredNS gene is used as a genomic fragment of influenza A virus in a methodwherein it is transferred to any desired influenza A virus strains orlive influenza vaccines by means of genetic reassortment. In thiscontext, it is preferred that the genetically engineered NS gene isestablished as a cDNA clone that can be transferred to any influenza Avirus strain or live influenza vaccine as a genomic fragment by means ofreverse genetics methods. For example, another vector such asAichi/NS-Nef belonging to the H3N2 subtype, but containing the samerecombinant NS-gene, can be obtained in this manner. In contrast tostrategies generating recombinant influenza viruses expressing foreignantigens in the context of HA or NA molecules, this approach enables afast generation of a set of non-crossreactive vectors for optimalboosting immunizations.

[0044] In a preferred embodiment of the invention the geneticallyengineered NS gene is designed for the expression of viral antigens,particularly for expression of the HIV-1 sequences of Nef or ELDKWA ofgp-41.

[0045] In another embodiment of the invention the genetically engineeredNS gene is rescued as a genomic fragment of influenza virus expressionof which contributes as or elicits a factor of protein kinase p-68 (PKR)over-expression and activation in infected cells.

[0046] In another embodiment of the invention the chimeric NS gene ispart of an attenuated (cold adapted) live influenza vaccine vectorwherein the genetically engineered NS gene is the main factor or anadditional factor of attenuation. It is particularly useful for themanufacture of safe and highly effective, influenza virus-based vaccinesincluding but not restricted to anti-HIV-1 vaccines, wherein thetransfected chimeric NS gene construct comprises gene sequences of nef,2A, and/or gp-41 or other viral antigens, for the induction of strongantibody and/or B- and T-cell immune responses.

[0047] The vaccine comprising an attenuated (cold adapted) liveinfluenza virus vector can be prepared in a suitable pharmaceuticalformulation and may be used for prophylactic immunizations as well asfor therapeutic vaccination, including induction of IFN release incombination with a stimulation of B- and T-cell response. In suchformulations influenza vectors might be used in combination with anyother vector expressing analogous antigens to ensure a maximum boostereffect. Thus, generating of attenuated influenza NS vectors offers thepossibility to obtain novel recombinant vaccines with nearly optimalbalance of safety and immunogenicity directed against a broad range ofpathogens.

[0048] In a particular embodiment of the invention the geneticallyengineered NS gene of an influenza A virus comprises a heterologousnucleotide insertion derived from the HIV-1 nef gene (nucleotides210-618 of the nef gene of the HIV-1 clone NL4-3) plus an insertion ofthe autocleavage sequence 2A (54 nucleotides) N-terminally to the HIV-1derived insertion at the position 400 of the NS protein. Elimination ofthe first 68 amino acids of the Nef protein was done to exclude domainscomprising the myristoylation site and other domains associated withpathogenic properties of the multifunctional HIV-1 Nef protein.

[0049] In further experiments the inventors found that influenza PR8/Nefvirus and influenza Aichi/Nef virus resulted in a high titer ofantibodies against the viral vector and a less but still significanttiter against the nef gene. The PR8/Nef virus is a PR8-124 virus withtruncated NS1 containing a 2A autocleavage sequence after aa position124 of the NS1 protein which additionally comprises a nef sequence (aa70-206 of HIV-1 Nef protein) following the autocleavage site. The Aichivirus is reassorted in that except for the NS gene all genes includingthe genes encoding the envelope proteins HA and NA originate from H3N2Aichi wild type virus, while the recombinant NS gene originates from thePR8/Nef virus. It was also observed that the PR8/Nef and Aichi/Nefviruses caused strong T-cell responses against the nef gene as well asthe viral vector. This experiment proved that it is possible to transferthe chimeric NS gene into another influenza virus strain to elicitessentially the same immune response. This finding is important as itallows to provide for possibilities to boost immunizations and to designseasonal influenza vaccines with varying immunogenic subtypes butconstant chimeric NS1 gene-based activity.

[0050] In another embodiment the invention provides for a method forgenerating recombinant influenza viruses by constructing a vectorcomprising a modified NS gene wherein the NS1 gene sequence is partiallyor entirely deleted or truncated, mixing said vector with lipids toallow self-assembling of lipid-DNA complexes and transfecting thelipid-DNA complexes into a desired continuous cell line, for example aVero or MDCK cell line, and selecting clones that stably integrate andreplicate the modified NS gene, which then are infected with any desiredinfluenza strain, and particularly, with an epidemic wild-type influenzastrain, to produce attenuated viral progeny containing said modified NSgene. In this method, the modified NS gene may further compriseinsertions of heterologous gene sequences coding, for instance, forother viral antigens or pathogens e.g. such as the ones disclosedherein.

[0051] It is another object of the present invention to provide a methodfor rapid vaccine manufacture comprising the steps of transforming acontinuous cell line to produce a desired synthetic viral gene,particularly a modified NS gene of influenza A virus wherein the NS1gene is partially or entirely deleted or truncated, infecting thetransformed cell line with a desired virus, particularly, with anepidemic wild-type influenza strain, to produce attenuated viral progenycontaining said modified NS gene, selecting attenuated recombinantviruses and multiplying such viruses under conditions suitable forefficient virus replication, preferably using interferon-deficientsubstrates, and combining harvested virus material with apharmaceutically acceptable carrier resulting in an anti-viral vaccine.In this method, the modified NS gene may further comprise insertions ofheterologous gene sequences coding, for instance, for other viralantigens or pathogens e.g. such as the ones disclosed herein. Furtherembodiments are defined in the dependent claims. In order that theinvention described herein may be more fully understood, the followingexamples are set forth. The examples are for illustrative purposes onlyand are not to be construed as limiting this invention in any respect.

EXAMPLE 1 Preparation of Recombinant Negative Strand Influenza A Viruses(“Reverse Genetics Method”)

[0052] The plasmid clones containing the nef sequence have been preparedon the basis of the existing plasmid clone of influenza NS genepUC19/NSPR (Egorov et al., 1998, J Virol 72/8, 6437-41). The nefsequence was inserted into the NS1 protein ORF, downstream of anadditional sequence: a protease recognition sequence P2A(NFDLLKLAGDVESNLG/P) derived from foot and mouth disease virus that isposttranslationally cleaved by an ubiquitous cellular protease (Mattionet al., 1996, J Ivrol 70(11), 8124-7; Percy et al., 1994, J Virol68(7),4486-92), so that the gp-41 molecule should be cleaved from theNS1 polypeptide and transported to the cell surface. The plasmid clonewas used for synthesis of chimeric RNA to be transfected into Vero cellsin order to rescue the recombinant influenza viruses. In the functionalmap of the engineered NS1 protein of the present invention (FIG. 1) itis indicated that the insertions are introduced after aa postion 124 andfollowed by a stop codon which has in effect that the remaining adjacentportion of the NS1 gene segment (inicuding the effector domain) restsuntranslated. From FIG. 2 it can be understood how the desired antigenicor otherwise heterologous sequences (e.g. gp-41 and IL-1β sequences) maybe arranged to yield immunogenic constructs that after transfection intoa suitable viral vector, preferably a cold adapted influenza virus,could form the basis of an effective vaccine against various infectiousdiseases. Analogously, FIG. 3 shows the arrangement of insertion ofaa70-206 of the Nef protein HIV-1 NL4-3 into the NS1 protein of therescued influenza transfectant PR82Anef(PR8/Nef).

[0053] In general, the experiments showed a tendency wherein the lengthof the heterologous insert or inserts was directly proportional to thedegree of attenuation of the resulting virus strain. Additionally, theimmunogenic potential of the expression products of larger insertsusually exceeded the one of smaller inserts. Therefore, it is preferredaccording to the present invention to make inserts encoding at leastabout 80 amino acids.

[0054] To create the chimeric Aichi/NS-Nef virus the RNA representingthe recombinant NS segment of the PR8/NS-Nef virus was introduced intothe genome of influenza A/Aichi/1/68 (H3N2) virus by a standard geneticreassortment performed on Vero cells utilizing rabbit polyclonal antiPR8 virus hyperimmune serum for selection. Genotyping of reassortantswas performed by RT-PCR amplification and comparative restrictionanalysis of cDNA copies derived from each genome segment.

EXAMPLE 2 Transfection of Recombinant Viruses in Vero Cells

[0055] Synthetic negative sense RNA have been derived from plasmidclones by T3 transcription in the presence of purified viral RNP. Verocells were 30 previously infected with the helper influenza virusreassortant strain 25A-1 (H1N1, (Egorov et al., 1994, Vopr Virusol39(5), 201-5) and then transfected with RNA complexes by DEAE-dextrantransfection (Egorov et al., 1998, J Virol 72(8), 6437-41; Luytjes etal., 1989, Cell 59(6), 1107-13). Rescued transfectant viruses have beenplaqued, purified on Vero cells 3 times, amplified on Vero cells andchecked for biological properties.

[0056]FIG. 10a-c show immunofluorescence of Vero cells infectedpreviously with recombinant PR8/Nef virus (MOI 0,01 in 10 a and 0,1 in10 b) and the wild type influenza PR8 virus (10c). 24 hrs followinginfection cells were trypsinized and fixed on cover slides with 100%acetone. After several wash steps in PBS the slides were incubated for40 min at 37° C. with a 1:50 dilution of anti Nef (aa179-195 epitope)mouse monoclonal antibody, then washed twice in PBS and incubated with1:100 dilution of goat anti mouse igG FITC conjugated antibody.

Example 3 Immunization of BALB/c Mice

[0057] Groups of three BALB/c mice each were immunized with 2-5×10⁵PFU/mouse of influenza viruses PR8/Nef, Aichi/Nef; PR8-124 and with4×10⁴ PFU/mouse of the PR8 wt as indicated in FIG. 5. Spleen cells fromimmunized mice were obtained 9 days later and used as effector cells inthe ELISPOT assay. FIG. 5 shows the number of IFN-gamma secreting cellsper 10⁶ spleen cells detected after immune spleen cells were incubatedin the presence of the EWRFDSRLAFHHVAREL peptide (Nef peptide),TYQRTRALVRTMGD peptide (NP peptide) or without peptide (w/o peptide).Results are expressed as average +/−SEM of duplicate cultures.

[0058] Groups of three BALB/c mice were immunized with 2-5×10⁵ PFU/mouseof influenza virus PR8/Nef, Aichi/Nef; PR8-124 and 2×10⁴ PFU/mouse ofthe PR8 wt as indicated in FIG. 6. Simple cells suspensions from thelymph nodes draining the respiratory tracts from immunized mice wereobtained 9 days later and used as effector cells in the ELISPOT assay(Power et al, J Immunol Methods 227:99-107): Briefly, threefold serialdilutions of cell populations derived from murine spleens, draininglymph nodes and the urogenital tracts were transferred to wells coatedwith anti IFN-γ mAb (R4-6A2; BD PharMingen). Cells were incubated for 22hours at 37° C. and 5% CO₂in DMEM medium containing 10% FCS, IL-2 (30U/ml), penicillin, streptomycin and 50μM 2-ME in the presence ofsynthetic peptides. A biotinylated anti IFN-γ mAb (XMG1.2; BDPharMingen) was utilized as a conjugate antibody, then plates wereincubated with streptavidin peroxidase (0.25 U/ml; Boehringer MannheimBiochemica). Spots representing IFN-γ secreting CD8⁺ cells weredeveloped utilizing the substrate 3-amino-9-ethylcarbazole (Sigma)containing hydrogen peroxide in 0.1 M sodium acetate, pH 5.0. The spotswere counted with the help of a dissecting microscope and results wereexpressed as the mean number of IFN-γ secreting cells±SEM of triplicatecultures. Cells incubated in the absence of synthetic peptides developed<10 spots/10⁶ cells. Since depletion of CD8⁺ cells resulted usuallyin>92% reduction of spot formation, cell separation was omitted in mostassays. FIG. 6 shows the number of IFN-gamma secreting cells per 106spleen cells detected after immune spleen cells were incubated in thepresence of the EWRFDSRLAFHHVAREL peptide (Nef peptide), TYQRTRALVRTMGDpeptide (NP peptide) or without peptide (w/o peptide).

[0059] From FIG. 4, which shows the replication of the chimericinfluenza/Nef viruses in mouse lower respiratory tracts, it can beunderstood that the PR8/Nef and Aichi/Nef viruses did not replicate inthat tissue, hence were strongly attenuated, while at the same they werehighly immunogenic to the mice causing strong T-cell and B-cell immuneresponses (as shown in FIGS. 5, 6 and 7). To characterize the insert(Nef peptide)-specific and vector (NP-peptide)-specific CD8⁺ T cellresponse female BALB/c mice were immunized once or twice i.n. withoutnarcosis with 10⁶ PFU per animal of the PR8/NS-Nef, Aichi/NS-Nef;PR8/NS-124 or PR8 w.t. virus.

[0060] Three BALB/c mice per group were immunized once or twice i.n. inthe absence of anesthesia with 10⁶ PFU/mouse of influenza PRS/NS-Nef,Aichi/NS-Nef, PR8/NS-124 or PR8 w.t. as indicated in FIG. 11. Thebooster immunization was performed 21 days after priming. The singlecell suspensions obtained 10 days after immunization from spleens ofmice were assessed for Nef peptide-specific (A) or NP peptide-specific(B) IFN-γ secreting CD8+T cells in an ELISPOT assay. FIG. 11 shows themean numbers of antigen-specific IFN-γ secreting cells±SEM of triplicatecultures.

[0061] Lymph nodes draining the respiratory tracts (mediastinal andretrobronchial lymph nodes) were collected 10 days after immunizationfrom immunized BALB/c mice as described in the FIG. 11. Single cellsuspensions were assessed for Nef peptide-specific (A) or NPpeptide-specific (B) IFN-γ secreting CD8⁺ T cells in an ELISPOT assay.FIG. 12 shows the mean numbers of antigen-specific IFN-γ-secretingcells±SEM of triplicate cultures. Three BALB/c mice per group wereimmunized twice i.n. in the absence of anesthesia with 106 PFU/mouse ofinfluenza PR8/NS-Nef, Aichi/NS-Nef or PR8/NS-124 virus as indicated inFIG. 13. The booster immunization was performed 21 days after priming.The single cell suspensions derived 10 days after the secondimmunization from digested urogenital tracts (vagina, cervix, uterinehorns and urethras) of immunized mice were assessed for Nefpeptide-specific (A) or NP peptide-specific (B) IFN-γ secreting CD8⁺ Tcells in an ELISPOT assay. FIG. 13 shows the mean numbers ofantigen-specific IFN-γ secreting cells±SEM of triplicate cultures.

[0062] Mice immunized once with either the PR8/NS-Nef or Aichi/NS-Nefvirus induced significant numbers of Nef peptide-specific CD8⁺ T cellsin single cell suspensions derived from spleens (139±4 spots inPR8/NS-Nef immunized mice; 137±39 spots in Aichi/NS-Nef immunized mice;FIG. 11B) and lymph nodes (173±23 spots in PR8/NS-Nef immunized mice;160±25 spots in Aichi/NS-Nef immunized mice; FIG. 12B). No relevant Nefpeptide-specific CD8⁺ T cell response was determined in bothcompartments of mice immunized with the PR8/NS-124 and PR8 w.t. viruses(the number of spots were always lower than 13; FIGS. 11B and 12B).

[0063] When vector (NP peptide)-specific CD8⁺ T cell responses werecompared, similar numbers of specific CD8⁺ spleen cells were found inall groups of mice tested (FIG. 11A). In contrast to the systemiccompartment (spleens), significant differences were obtained in themucosa-associated respiratory lymph nodes. The replication competentPR8/NS-124 virus induced a markedly higher frequency of NP-peptidespecific CD8⁺ T cells whereas recombinant influenza/NS-Nef viruses andthe pathogenic PR8 w.t. virus induced lower magnitudes of NPpeptide-specific CD8⁺ T cells (FIG. 12A).

[0064] In FIG. 7, which displays Nef-specific serum IgG immune responsesof mice immunized with recombinant influenza/Nef viruses, the B-cellimmune responses following 2nd and 3rd immunizations have beendemonstrated to be strongest in the case where the mice have beenimmunized twice by PR8/Nef followed by a third immunization withAichi/Nef, while the response was less prominent with twiceimmuniziations using the PR8-124 virus.

[0065] Further, a group of mice was primed i.n. with the PR8/NS-Nefvirus and boosted 21 days later with the Aichi/NS-Nef virus. Anothergroup of mice was immunized with the same viruses but in the reverseorder. Data shown in FIG. 11 and 12 indicate that the sequence in whichthe respective recombinant vectors were used for priming and boostingappeared to be crucial, since it was consistently observed that primingwith the Aichi/NS-Nef (H3N2) followed by boosting with the PR8/NS-Nef(H1N1) induced a significantly lower number (approximately the range ofthe primary CD8⁺ T cell response) of the Nef peptide-specific and NPpeptide-specific CD8⁺ T cells in spleens and draining lymph nodes whencompared with the reverse order of immunization. A strong secondaryantigen specific CD8⁺ T cell response was detected in both of thecompartments tested after priming the mice with the recombinantPR8/NS-Nef (H1N1) vector followed by a boost using the H3N2 subtypeAichi/NS-Nef vector. In this case, Nef- and NP peptide-specificsecondary responses were approximately 1.5 to 3 times higher than aftera single immunization (FIGS. 11 and 12).

[0066] Single cell suspensions derived from the urogenital tracts wereobtained from immunized mice. Two immunizations were necessary beforesignificant numbers of Nef peptide-specific CD8⁺ T cells could bedetected. The strongest Nef peptide specific CD8⁺ T cell response wasdetected when mice were primed i.n. with PR8/NS-Nef (H1N1) virus andsubsequently boosted with the Aichi/NS-Nef (H3N2) virus (342±18 IFN-γSC/10⁶ cells; FIG. 13B). This immunization protocol was also found toinduce the strongest NP peptide-specific CD8⁺ T cell response (FIG.13A).

[0067] As described for the detection of T-cell responses mice wereutilized to assess the Nef-specific serum antibody response. Mice wereprimed i.n. either with 106 PFU/ml of the PR8/NS-Nef (H1N1) orAichi/NS-Nef (H3N2) virus and were boosted three weeks later with thesame vector. The third immunization was performed following three moreweeks utilizing the vector of the different subtype. The control groupwas immunized three times with the PR8 w.t. virus. The reactivities ofserum samples (obtained two weeks after the third immunization) with theGST-Nef fusion peptide were determined by ELISA and are shown in FIG.14. Nef-specific antibodies were detected only in groups of mice whichhad been successively immunized with H1N1 and H3N2 vectors (FIG. 14).The highest level of Nef-specific IgG was detected in mice immunizedtwice i.n. with 106 PFU of the PR8/NS-Nef (H1N1) virus and boosted with106 PFU of the Aichi/NS-Nef (H3N2) virus as compared with the controlgroup which had been immunized three times i.n. with PR8 w.t. virus(FIG. 14).

[0068] Both influenza virus vectors (PR8/NS-Nef and Aichi/NS-Nef) werecompletely attenuated in mice, since no viral titers could be detectedin mouse respiratory tissues. These attenuated phenotypes of bothrecombinant viruses indicate that introduction of additional amino acidsdownstream of the position 125 of the NS1 protein can affect somefunction of the NS1 protein since PR8/NS-124 virus encoding the samesize of the NS1 protein grew efficiently in mouse respiratory tracts(FIG. 4). The low efficiency of the 2A site to cleave Nef antigen fromthe N-terminal part of NS1 protein, especially at the late stage ofinfection, might be responsible for additional attenuation, although adirect effect of the Nef polypeptide interacting with some intracellularcomponents can not be excluded.

[0069] The data indicate that completely attenuated recombinantinfluenza/NS-Nef viruses are capable to induce a primary CD8⁺ T cellresponse directed to the inserted Nef polypeptide in spleens and inlymph nodes draining the respiratory tracts of mice immunized i.n.without anesthesia. At the same time, vector (NP peptide)-specific CD8⁺T cell responses in spleens and draining lymph nodes of mice immunizedi.n. either with the PR8/NS-Nef or Aichi/NS-Nef vector were in the rangeof those induced by the virulent PR8 w.t. virus although the strongestNP-peptide specific CD8⁺ T cell response detected in the draining lymphnodes was found in mice immunized with the PR8/NS-124 virus efficientlyreplicating in the lungs.

[0070] The results indicate that it is possible to achieve a similareffect utilizing influenza vectors belonging to different antigenicsubtypes. Importantly, influenza virus vectors are capable to induce aCD8⁺ T cell response circumventing the pre-existing immunity caused by adifferent influenza virus subtype.

[0071] The immunogenic potential of attenuated influenza/NS-Nef vectorsmight be explained by the fact that viruses containing truncated formsof the NS1 protein induce high levels of type 1 interferons in vivo.Nef-expressing vectors as well as PR8/NS-124 virus induced significantlyhigher levels of type 1 interferons in serum following immunization ofmice if compared with the corresponding w.t. parent viruses (data notshown).

EXAMPLE 4 Plaque Reduction Assay

[0072] MDCK cells were treated for 24 h with a supernatant from MDCKcells infected by deINS virus (same construct as disclosed in WO99/64571, i.e., containing entire NS1 deletion) as a known potent IFNalfa/beta inducer (FIG. 258). The content of the IFNalfa/beta wasestimated to 100 U following an overnight treatment with pH 2. Theresults in FIG. 8 are given in log of plaque forming units (PFUreflecting the differences in viral titers on IFNalfa/beta treated anduntreated cells.

EXAMPLE 5 Interferon Induction

[0073] MDCK cells were infected for 24 h with 5 MOI of differentinfluenza viruses as outlined in FIG. 9. The supernatants were treatedovernight with pH 2 at 4° C. for virus inactivation. Treatedsupernatants were adjusted to pH 7.4 with. 1 N NaOH. Twofold serialdilutions of these supernatants were added to MDCK monolayers for 24 hrsand 50 PFU of the vesicular stomatitis virus (VSV) were then added perwell. The results represent the dilution of the supernatant at which VSVplaque formation was reduced by 50%.

EXAMPLE 6 A method for Accelerated Production of Recombinant Viruses andAnti-Viral Vaccines

[0074] a) Generating Recombinant Cell Lines Producing Modified NS Gene:

[0075] A plasmid vector was constructed according to the schematicrepresentation in FIG. 15. The NS gene was cloned into the backbone of apS65T-C1 vector (Clontech), using the CMV promotor to initiatetranscription. Inserting NS in reverse orientation (3′ end towards CMVpromotor) leads to the transcription of minus sense RNA. Transcriptionis terminated by a hepatitis delta virus (HDV) sequence that comprises aself-cleaving RNA site. Hence, this vector contains an influenza A NSgene where the cassette of the multiple stop codons is introduced at ntposition 140 in a manner such that translation of this gene (readingframe 3) leads to a truncated form of the NS1 protein (comprising only38 amino acids). It was found that influenza A viruses expressing such ashort NS1 protein are highly attenuated in animals (Egorov et al. 1998,J Virol. 72, 8, p 6473). Sequence of HDV (85 nt):TGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCCGAGGGGACCGTCCCCTCGGTAATGGCGAATGGGAC Sequence of PR8NS38: (906 nt)AGCAAAGCAGGGTGACAAAGACATAATGGATCCAAACACTGTGTCAAGCTTTCAGGTAGATTGCTTTCTTTGGCATGTCCGCAAACGAGTTGCAGACCAAGAACTAGGTGATGCCCCATTCCTTGATCGGCTTCGCCGAGTGAATAACTAGCTGAATCAGAAATCCCTAAGAGGAAGGGGCAGCACCCTCGGTCTGGACATCGAGACAGCCACACGTGCTGGAAAGCAGATAGTGGAGCGGATTCTGAAAGAAGAATCCGATGAGGCACTTAAAATGACCATGGCCTCTGTACCTGCGTCGCGTTACCTAACTGACATGACTCTTGAGGAAATGTCAAGGGACTGGTCCATGCTCATACCCAAGCAGAAAGTGGCAGGCCCTCTTTGTATCAGAATGGACCAGGCGATCATGGATTAAGAACATCATACTGAAAGCGAACTTCAGTGTGAATTTTGACCGGCTGGAGACTCTAATATTGCTAAGGGCTTTCACCGAAGAGGGAGCAATTGTTGGCGAAATTTCACCATTGCCTTCTCTTCCAGGACATACTGCTGAGGATGTCAAAAATGCAGTTGGAGTCCTCATCGGGGGACTTGAATGGAATGATAACACAGTTCGAGTCTCTGAAACTCTACAGAGATTCGCTTGGAGAAGCAGTAATGAGAATGGGAGACCTCCACTCACTCCAAAACAGAAACGAGAAATGGCGGGAACAATTAGGTCAGAAGTTTGAAGAAATAAGATGGTTGATTGAAGAAGTGAGACACAAACTGAAGATAACAGAGAATAGTTTTGAGCAAATAACATTTATGCAAGCCTTACATCTATTGCTTGAAGTGGAGCAAGAGATAAGAACTTTCTCGTTTCAGCTTATTTAGTAATAAAAAACACCCTTGTTT CTACT

[0076] This plasmid vector was used for transfection in order totransform Vero cells. Before transfection, cells were seeded inappropriate cell culture flasks (e.g. 25 cm) and incubated at 37° C.until having reached about 50% confluence. To improve the transfectionefficiency the cationic lipid reagents (lipofectin, lipofectamine 2000)may be preincubated in medium without serum prior to mixing it with theplasmid-DNA. Accordingly, 2-6 μl cationic lipid reagent were diluted in100 μl OPTI-MEM I serum free transfection medium and incubated 30 min atroom temperature. Meanwhile 1-2 μg DNA were diluted in 100 μl OPTI-MEMI, then mixed with the lipid solution and incubated 15 min at roomtemperature. While DNA-lipid complexes formed, the cells were washedtwice with serum-free transfection medium to remove residual proteins.The transfection cocktail was diluted to a total volume of 1 ml usingtransfection medium and added to the cells. Cells were incubated at 37°C. with 7% CO₂ from 5 to 8 h. Thereafter cell culture supernatant wasremoved and cells were fed with normal culture medium. 24 hours posttransfection stable transfectants were selected by addition of selectionmedium containing geneticin sulfate G418 (400 μg/ml). Three weeks laterstable transfectants appeared which were subcloned by limitingdilutions.

[0077] Several subclones were tested for their ability to rescue the tshelper virus (25A-1 mutant, which is a reassortant virus wherein the NSgene responsible for the ts phenotype originates from cold-adaptedinfluenza strain A/Leningrad/134/47/57, and the remaining genesoriginate from PR8 virus; Egorov et al., 1994, Vopr. Virusol.39:201-205) at a temperature of 40° C. For that purpose, the cells wereinfected with the 25A-1 mutant and incubated at 40° C. for 72 hours.Those subclones yielding viral progeny that contained influenza virusescarrying the recombinant NS gene were selected and further multipliedunder conditions allowing for efficient cell growth.

[0078] b) Recombinant Influenza Virus and Vaccine Production:

[0079] Transformed cells expressing a modified NS gene segmentcomprising a truncated NS1 gene with or without insertions, preferablyas described hereinbefore, are infected with a desired influenza virus,particularly with a wildtype epidemic virus, under conditions asdescribed above and incubated to allow for the development of viralprogeny wherein the wildtype NS gene is replaced by the recombinantmodified NS gene supplied by the transformed host cells. The viral yieldcan be cloned by plaquing methods (e.g. negative colonies under agaroverlay) on a normal Vero cell line and each viral colony can bescreened for the presence of the modified recombinant NS gene by theRT-PCR method and/or by other methods (depending on how the gene wasmodified). Positive viral plaques are then purified by further plaquingpurification steps. Finally, the recombinant influenza strains arehighly attenuated because of the truncated NS protein. They serve asvaccine candidates and are multiplied preferably using IFN deficientsubstrates such as Vero cells, young chicken embryos (less than 10 daysold) and the like, for rapid manufacture of highly attenuated liveinfluenza vaccines.

[0080] It is pointed out, however, that the method described in thisExample is just one way to exemplify the underlying general concept ofpreparing and establishing mammalian cell lines, particularlyimmortalized or continuous cell lines, that after transformation withany desired viral gene sequences stably integrate and express thesesequences, and thus allow for relatively simple and rapid design andmanufacture of reasserted, recombinant viruses of whatever origin.ABBREVATIONS aa amino acid CMV cytomegalovirus CTL cytotoxicT-lymphocyte gp glycoprotein HA haemagglutinin IFN interferon Igimmunoglobulin IL interleukin i.n. intranasally mAb monoclonal antibodyMHC major histocompatibility complex NA neuramidase nef negative factorof HIV NP nucleoprotein NS non-structural ORF open reading frame RNPribonucleoprotein SIV Simian immunodeficiency virus ts temperaturesensitive v viral VSV vesicular stomatitis virus wt wild type

We claim:
 1. A recombinant NS gene of an influenza A virus comprising afunctional RNA binding domain and a gene sequence modification afternucleotide position 400 of the NS1 gene segment, counted on the basis ofinfluenza A/PR/8/34 virus, wherein the modification bars transcriptionof the remaining portion of the NS1 gene segment.
 2. The recombinant NSgene according to claim 1, wherein the modification comprises deletions,insertions, or a shift of the open reading frame.
 3. The recombinant NSgene according to claim 1, wherein the modification comprises aninsertion of at least one sequence selected from the group consisting ofan autocleavage site 2A, the nef gene from HIV-1, the sequences encodingthe ELDKWA or ELDKWAS epitopes of gp41 of HIV-1, the sequence encodingIL-1β or a part thereof, a leader sequence, and an anchor sequence. 4.The recombinant NS gene according to claim 3, wherein insertion encodesa sequence of at least 80 amino acids.
 5. A genetically engineered,preferably cold adapted, influenza virus comprising a modified NS geneas defined in any one of claims 1 to
 4. 6. The influenza virus accordingto claim 5, having an IFN inducing phenotype.
 7. The influenza virusaccording to claim 5 or 6, which is not IFN sensitive.
 8. A vaccinecomprising at least one virus, preferably a mixture of differentviruses, as defined in any one of claims 5 to 7, in a suitablepharmaceutical formulation.
 9. The vaccine according to claim 8, forprophylactic or therapeutic application against a viral infection,preferably against influenza or HIV-1 infection.
 10. A method for themanufacture of recombinant influenza viruses comprising the steps of:transforming a mammalian cell, preferably a continuous cell line, with aDNA vector comprising a modified NS gene segment wherein the NS1 genesequence is partially or entirely deleted or truncated, selectingtransformed cells that express the modified NS gene segment, infectingthe selected cells with a desired influenza virus, preferably awild-type epidemic strain, incubating the infected cells to allow forthe development of viral progeny containing the modified NS genesegment, and selecting and harvesting said viral progeny containing themodified NS gene segment.
 11. The method according to claim 10, whereintransformation of the mammalian cell is accomplished comprising mixingsaid vector with lipids to allow for a self-assembly of the lipid andthe DNA to form lipid-DNA complexes, and incubating the mammalian cellsin the presence of said lipid-DNA complexes resulting in an uptake ofthe complexes into the cells, and preferably into the cell nucleus. 12.The method according to claim 10 or 11, wherein the DNA vector is atranscription system for minus sense influenza RNA.
 13. The methodaccording to any one of claims 10 to 12, wherein said viral progenycontaining the modified NS gene segment is further combined with apharmaceutically acceptable carrier for use as an attenuated influenzalive vaccine.
 14. A live attenuated influenza virus vaccine comprisingat least one genetically engineered, preferably cold adapted, influenzavirus as defined in claim 5, preferably a mixture thereof, obtainable ina method according to any one of claims 10 to 13.